Otilonium and pinaverium trigger mitochondrial-mediated apoptosis in rat embryo cortical neurons in vitro.

In the frame of a repositioning programme with cholinergic medicines in clinical use searching for neuroprotective properties, we surprisingly found that spasmolytic antimuscarinics otilonium and pinaverium exhibited neurotoxic effects in neuronal cultures. We decided to characterize such unexpected action in primary cultures of rat embryo cortical neurons. Neurotoxicity was time- and concentration-dependent, exhibiting approximate EC50 values of 5 μM for both drugs. Seven antimuscarinic drugs endowed with a quaternary ammonium, and another 10 drugs with different cholinergic activities, carrying in their molecule a ternary ammonium did not exhibit neurotoxicity. Both drugs caused a concentration-dependent blockade of whole-cell inward currents through voltage-activated calcium channels (VACCs). Consistent with this, they also blocked the K+ -elicited [Ca2+ ]c transients. Neither antioxidant catalase, glutathione, n-acetylcysteine, nor melatonin protected against neurotoxicity of otilonium or pinaverium. However cyclosporine A, a blocker of the mitochondrial permeability transition pore, prevented the neurotoxic effects of otilonium and pinaverium monitored as the fraction of cells undergoing apoptosis. Furthermore, the caspase-9 and caspase-3 inhibitor Ac-LEHD-CHO mitigated the apoptotic neuronal death of both drugs by around 50%. Data are compatible with the hypothesis that otilonium and pinaverium elicit neuronal death by activating the intrinsic mitochondrial-mediated signaling pathway of apoptosis. This may have its origin in the mitigation of Ca2+ entry and the uncoupling of the Ca2+ -dependent generation of mitochondrial bioenergetics, thus causing the opening of the mitochondrial mPTP to elicit apoptotic neuronal death.